Abstract

Background:Gardenia jasminoides is a natural plant, it has many biological activities. Its ability of cancer cell apoptosis needs to be researched.

Methods: In this study, the important biological activity materials were determined by high efficiency liquid chromatography. The in vitro anticancer effects were measured by MTT and RT-PCR assays in HepG2 human hepatoma cells.

Conclusion:Gardenia jasminoides had a strong anticancer effect through its apoptosis inducing abilities in hepatic cancer; it could be used for the treatment as a medicine or health product in daily human life for good liver health.

Keywords

Introduction

Gardenia jasminoides is dried mature fruit of Gardenia jasminoides jasminoides Ellis, which belongs to the family
Rubiaceae [1]. It has many common beneficial effects for daily
life, calm fret, diuresis, cold blood and detoxication. However,
it has detumescence analgesic effect for topical use [2]. It
includes many kinds of chemicals, such as iridoids, diterpenes
(crocins), organic acids, flavonoids, coumarins, naphtha,
saponins, lignanoids and polysaccharides. Meanwhile, the
study emphasize that Gardenia jasminoides has a number of
ingredients with strong physiological activities, mainly iridoids
and crocins [3]. Genipin gentiobioside, gardenoside, crocin-1
and crocin-2 are important contents of Gardenia jasminoides,
these contents have anticancer effects [3], gardenoside has in vitro anticancer effects in HepG2 liver cancer cells [4], both
crocin-1 and crocin-2 could treat liver cancer [5].

As one of the most common malignant tumors, liver cancer is
extremely severe and had a great impact on human health,
which mostly common in males at the age of 40-50 [6]. As
compared to other cancers mortality and morbidity is high,
whereas survival rate is short, so it does severe harm to human
body. The attack of liver cancer is related to various factors
such as virus, chemical compounds, genetics and diet [6,7].
Among these, the main factors that promote liver cancer are
alcohol, moldy food, food containing nitrosamines, foods
which are lack of trace element selenium [8]. The bioactive
elements in Gardenia jasminoides can adjust the harm to
human body caused by unhealthy lifestyle, while they also
have certain effects on many kinds of cancer cells [2,9]. Studies have shown that geniposide has obvious inhibiting
effect on many kinds of cancer, especially pancreatic cancer
[10,11]. In addition, modern pharmacological studies have
shown that crocin has obvious anti-cancer function, especially
inhibiting the cancer cells of cervical cancer, leukemia, bladder
cancer, colon cancer, rectal cancer and so on [12-14].

Cancerous tumor form due to the inhibition of apoptosis [15].
It is generally seen that malignant tumor cells are caused by
wild growth and excessive proliferation. At the same time, a
series of oncogenes and proto-oncogenes in cancer cells are
activated and present expressing state. The activation of these
genes has close relationship with the development of tumor
[16]. Among cancer genes, some belong to the growth factor
genes, while others belong to the growth factor receptor genes.
The activation and expression of these genes directly stimulate
the growth of tumor cells, and these cancer genes and their
expressed products are an important regulatory factors of cell
apoptosis, as many cancer genes block the apoptosis process of
tumor cells and after expression, lead to the increase of tumor
cells [17]. Therefore, controlling tumors through controlling
cell apoptosis, its mechanism is to rebuild tumor cell
transformation system of apoptosis and activate the expression
of death gene in cancer cells [18]. The important effective
ingredient crocin in Gardenia jasminoides can obviously
inhibit the multiplication of human leukemia cell line HL-60
and block cell cycle G0/G1 [19]. The apoptosis-inducing effect
may be related to inhibit the expression of Bcl-2 genes and
active the expression of Bax genes. Bcl-2 family genes are
important cell apoptosis genes [20], and the cancer cell
apoptosis-inducing effect of many bioactive substances in Gardenia jasminoides may be the key to the anti-cancer effect
of Gardenia jasminoides. At the same time, the associative
function of different substances may enhance the effect of Gardenia jasminoides. Therefore, this research study is to
investigate the antioxidant effect of Gardenia jasminoides in
vitro to clarify anti-cancer mechanism of Gardenia jasminoides by observing its cancer cell apoptosis-inducing effect.

Materials and Methods

Preparations of Gardenia jasminoides extract

Gardenia jasminoides was purchased in a local market (Enshi
City, Hubei Province, China) and stored at -80°C prior to being
freezedried to produce a powder. A 20fold volume of 70%
methanol was added to the powdered Gardenia jasminoides and then extracted by sonic extract (power 250W, frequency 40
kHz). The methanol extract was evaporated using a rotary
evaporator (N1100; Eywla, Tokyo, Japan), concentrated and
then dissolved in dimethyl sulfoxide (DMSO; Amresco, Solon,
OH, USA) to adjust to the stock concentration (20%) [21].

Growth of HepG2 and L-02 cells by different concentrations of Gardenia jasminoides extract treatment were determined by
the MTT assay. Different concentrations of Gardenia jasminoides (50, 100, 200 and 400 μg/mL) and the caspase
inhibitor Z-VAD-FMK (10 μg/mL) were added to the RPMI
1640 medium. HepG2 cancer cells were seeded in 96 well
plates at the density of 1 × 105 cells/mL in each well for 100
μL, and then the cells were incubated at 37 °C in 5% CO2.
After the cancer cells were cultured for 24 h, the culture
medium was aspirated and discarded from each well, and then
the new culture medium with different concentrations of Gardenia jasminoides was added to each well. The cells were
continuing incubated for 48 h and the culture medium was
aspirated and discarded in each well again. Then the culture
medium contained 5 mg/mL MTT solution (Amresco; Solon,
OH, USA) was added in each well and cultured for 4 h.
Following removal of the MTT solution culture medium, 100
μL of DMSO was added to each well and mixed for 30 min.
Subsequently, the absorbance of each well was measured with
an enzyme-linked immunosorbant assay (ELISA) reader
(model 680; Bio-Rad; Hercules, CA, USA) at 540 nm [23].

RT-PCR assay

Total RNA from different groups HepG2 cells were isolated
using Trizol reagent (Invitrogen; Carlsbad, CA, USA)
according to the manufacturer’s recommendations. The HepG2
cells RNA were digested with RNase-free DNase (Roche;
Basel, Switzerland) for 15 min at 37°C and purified using the RNeasy kit (Qiagen; Hilden, Germany) according to the
manufacturer’s protocol. cDNA in cancer cells were
synthesized from 2 μg of total RNA by incubation at 37°C for l
h with avian myeloblastosis virus reverse transcriptase (GE
Healthcare; Little Chalfont, United Kingdom) with random
hexanucleotides according to the manufacturer’s instruction.

Sequences of primers used to specifically amplify the genes of
interest are shown in Table 1. Amplification was performed in
a thermal cycler (Eppendorf; Hamburg, Germany). The
polymerase chain reaction (PCR) products were separated in
1.0% agarose gels and visualized with ethidium bromide
staining [23].

Gene Name

Sequence

Caspase-3

Forward: 5′-CAA ACT TTT TCA GAG GGG ATC G-3′

Reverse: 5′-GCA TAC TGT TTC AGC ATG GCA-3′

Caspase-8

Forward: 5′-CCC CAC CCT CAC TTT GCT-3′

Reverse: 5′-GGA GGA CCA GGC TCA CTT A-3′

Caspase-9

Forward: 5′-GGC CCT TCC TCG CTT CAT CTC-3′

Reverse: 5′-GGT CCT TGG GCC TTC CTG GTA T-3′

Bax

Forward: 5′-AAG CTG AGC GAG TGT CTC CGG CG-3′

Reverse: 5′-CAG ATG CCG GTT CAG GTA CTC AGT C-3′

Bcl-2

Forward: 5′-CTC GTC GCT ACC GTC GTG ACT TGG-3′

Reverse: 5′-CAG ATG CCG GTT CAG GTA CTC AGT C-3′

Bcl-xL

Forward: 5′-CCC AGA AAG GAT ACA GCT GG-3′

Reverse: 5′-GCG ATC CGA CTC ACC AAT AC-3′

XIAP

Forward: 5′-CCG TGC GGT TGC TTT AGT TGT C-3′

Reverse: 5′-ATG GCA GGG TTC CTC GGG TAT-3′

cIAP-1

Forward: 5′-TGAGCATGCAGACACATGC-3′

Reverse: 5′-TGACGGATGAACTCCTGTCC-3′

cIAP-2

Forward: 5′-AATGGAAGATAGCACGAT-3′

Reverse: 5′-AGAAAGGCTGGAGTAAGA-3′

Survivin

Forward: 5′-CTT TCT CAA GGC CCA CCG CAT CT-3′

Reverse: 5′-GCA CTT TCT CCG CAG TTT CCT C-3′

TRAIL

Forward: 5′-GGA ACC CAA GGT GGG TAG AT-3′

Reverse: 5′-TCT CAC CAC ACT GCA ACC TC-3′

Fas

Forward: 5′-GAA ATG AAA TCC AAA GCT-3′

Reverse: 5′-TAA TTT AGA GGC AAA GTG GC-3′

FasL

Forward: 5′-GGA TTG GGC CTG GGG ATG TTT CA-3′

Reverse: 5′-TTG TGG CTC AGG GGC AGG TTG TTG-3′

p53

Forward: 5′-GCT CTG ACT GTA CCA CCA TCC-3′

Reverse: 5′-CTC TCG GAA CAT CTC GAA GCG-3′

p21

Forward: 5′-CTC AGA GGA GGC GCC ATG-3′

Reverse: 5′-GGG CGG ATT AGG GCT TCC-3′

NF-κB

Forward: 5′-CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3′

Reverse: 5′-CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3′

IκB-α

Forward: 5′-GCT GAA GAA GGA GCG GCT ACT-3′

Reverse: 5′-TCG TAC TCC TCG TCT TTC ATG GA-3′

GAPDH

Forward: 5′-CGG AGT CAA CGG ATT TGG TC-3′

Reverse: 5′-AGC CTT CTC CAT GGT CGT GA-3′

Table 1. Sequences of reverse transcription-polymerase chain reaction primers were used in this study.

Statistical analysis

All experiments were determined three times and all the data
were presented as mean ± standard deviation (SD). Differences
between the mean values for individual groups were assessed
with one-way analysis of variance (ANOVA) with Duncan's
multiple range test. P<0.05 was considered to indicate a
statistically significant difference. SAS version 9.2 (SAS
Institute Inc., Cary, NC, USA) was used to conduct the
statistical analyses.

Results

The main constituent of Gardenia jasminoides

In preliminary experiments, observe extraction solvents (n-hexane,
ethanol and methanol), extraction methods (cold
leaching, ultrasonic extraction and reflux), extraction time (15,
30, 45, 60 mins) as well as the amount of extraction solvents
(50, 75, 100, 125 times). Based on the principles of efficiency,
convenience and saving, the extraction method was to use 0.2 g
sample powder with 20 mL 70% methanol for 30 mins
ultrasonic extraction. Respectively full scan reference substance solutions of all four index components, the result
showed that the maximum absorption wavelength of genipin
gentiobioside and geniposide were both at 238 nm, while the
maximum absorption wavelength of crocin 1 and crocin 2 were
both at 440 nm. Peak time of the first two components was
from 2 to 5 mins, while peak time of the rest two components
was from 5 to 8 mins. As a result, adding wavelength
switching events makes the detection of wavelength switch
automatically at the 5th minute, achieving detection of the four
indicators at the same time.

Figure 2. Effect of different concentrations of Gardenia jasminoides on the growth of HepG2 human hepatoma cells (A) and human normal hepatic cell line L-02 (B). Cell viability was evaluated with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.

At the concentrations ranging from 0 to 600 μg/mL of Gardenia jasminoides, HepG2 cells were decreased by the
sample in a concentration-dependent manner, at the
concentration of 600 μg/mL, the survival rate of the HepG2
cells treated with Gardenia jasminoides reached 0%, but the
normal hepatic cells L-02 were not significantly reduced.
Consequently, 0-400 μg/mL Gardenia jasminoides were safe
concentrations for normal human hepatic cells, and 50, 100,
200 and 400 μg/mL selected for subsequent human cancer cell
experiments.

a-f, A-E Mean values with different letters in the same column are significantly different (P<0.05) according to Duncan’s multiple-range test. Z-VAD-FMK treatment: 400 µg/mL Gardenia jasminoides + 10 µg/mL Z-VAD-FMK.

Table 4. Growth inhibition of HepG2 human hepatoma cells by different concentrations of Gardenia jasminoides as evaluated by an MTT assay.

Gene expression of caspase family

The caspase-3, caspase-8 and caspase-9 mRNA expressions
were determined by RT-PCR assay (Figure 3). The untreated
HepG2 cancer cells (control) had the lowest caspase-3,
caspase-8 and caspase-9 mRNA expression. After treated with different concentrations of Gardenia jasminoides, the
caspase-3, caspase-8 and caspase-9 mRNA expressions were
raised, these expressions were significantly different (P<0.05)
from control cancer cells, and 400 μg/mL has the highest
concentration of Gardenia jasminoides treated cells had the
strongest caspase-3 (3.23 folds of control), caspase-8 (5.19
folds of control) and caspase-9 (4.57 folds of control) mRNA
expressions.

The Fas expressions in Gardenia jasminoides treated HepG2
cells were stronger than control cancer cells (Figure 6), the
expressions of 50, 100, 200, 400 μg/mL Gardenia jasminoides treated cells were 1.37, 1.47, 1.73, 1.80 folds than the control
cells. The FasL expressions in Gardenia jasminoides treated
group cells were not have a meaningful difference (P<0.05),
but the FasL expressions in Gardenia jasminoides treated cells
were (P<0.05) raised remarkably as compared to the control cells. Fas/FasL ratio which (P<0.05) increased after treated
with Gardenia jasminoides in HepG2 cells, and the ratio of 400
μg/mL concentration treatment were significantly high in 1.46.

Discussion

Gardenia jasminoides has many physiological activities, and
the antioxidant activities could help Gardenia jasminoides treat
cancer [24,25]. Oxygen free radicals are one of the main causes
of cell damage, aging and some diseases, Gardenia jasminoides contains many flavanoids, these flavanoids make a
strong antioxidant activity, this effect might help Gardenia jasminoides has anticancer effects [25,26]. Gardenia jasminoides only had inhibitory effects in HepG2 cancer cells,
but had no effects in normal hepatic cells L-02 at 0-400 μg/mL,
its anticancer effects were determined at these concentrations
in this study. Caspase-3 is a kind of proteolytic enzyme
mediating apoptosis that makes it a key effect enzyme in
caspase, which is an important effector molecule to perform
apoptosis. It is widely expressed in normal human tissues and a
variety of tumor tissues [27]. Studies have shown that the
activation of Caspase-3 protease is closely related to the
apoptosis of liver cancer cells. The activation of Caspase-3
exists in two different ways, including caspase-8 or caspase-10
dependent way and caspase-9 dependent way [28,29].
Caspase-3 is the core protease to trigger cell apoptosis protease
cascade reaction, which plays a very important role. Caspase-3
is activates the liver cancer cell apoptosis induced by a variety
of factors, Therefore, inhibiting the activity of caspase-3 can
prevent apoptosis of liver cancer cell [30]. Z-VAD-FMK is a
caspase inhibitor, Z-VAD-FMK treatment could determine
whether the anticancer effects of drug from its caspase change
ability [31]. In this study, after Z-VAD-FMK treatment, the
anticancer effects of Gardenia jasminoides were reduce; these
results showed that the anticancer of Gardenia jasminoides might through its caspase decreasing effects. Bcl-2 family
including gene products Bcl-2 and Bcl-xL can prevent
apoptosis, while gene products such as Bax fight against the
above-mentioned genes usually promote cell apoptosis. Bcl-2
family plays an important regulating role in the activation
process of caspase-3 [32]. Anti-apoptosis Bcl-2 family member
Bcl-xL inhibits the oligomeric function of Apaf-1 molecules,
making them dysfunctional. Mainly existing on the outer
membrane of mitochondrion, anti-apoptotic Bcl-2 family
inhibits the activation of pro caspase-9 by inhibiting the release
of cytochrome C from mitochondrion, thus inhibiting the
activation of caspase-9 which Apaf-1 depends on [33]. Pro-apoptosis
Bcl-2 family members Bak and Bik can inhibit
contact between Apaf-1 and Bcl-xL, thus promoting the
activation of caspase which Apaf-1 depend on. Bid belongs to
Bcl-2 family, which causes apoptosis. Studies have found that
caspase-8 can crack Bid, and cracked Bid can react with Bcl-xL
which exists on the surface of mitochondrion to induce
mitochondrion to release cytochrome C, which activates
caspase-9. Caspase-8 and caspase-9 are connected by Bid. This
kind of crosstalk can amplify the process of caspase activation
[34].

As one TNF family member, APRIL can promote the
proliferation and apoptosis of tumor cells by interfering with
apoptosis signaling pathway or regulating expression of related
anti-apoptotic genes in autocrine or paracrine way. APRIL
shows high expression in many kinds of malignant tumor
tissues, while only a small amount expresses in normal tissues
[35]. As an important member in IAPs family, XIAP has
special BIR structure, so it can combine with caspase and
inhibit its activity, which promotes the occurrence and
development of tumors. XIAP shows high expression in many
tumor tissues and cells [36]. Survivin is a kind of strong
apoptosis-inhibiting factor, and many studies have shown that
high expression of survivin inhibits apoptosis induced by Fas,
Bax, caspases, tumor necrosis factors, as well as anti-cancer
drugs [37]. At the same time, many studies have also shown
that survivin may be regarded as an indicator for diagnosing
liver cancer and predicting metastasis, recurrence and
prognosis of liver cancer, so it is a suitable target for tumor
biological treatment [38-40].

As a kind of transcription factor, NF-κB fights against
apoptosis mainly by regulating downstream anti-apoptotic
genes. NF-κB can increase apoptosis functional elements, such
as Bcl-2 family and IAP family, etc [41]. Bcl-2 is a recognized
anti-apoptotic protein, as many tumor cells show high
expression of Bcl-2 and play an anti-apoptotic role through
Bcl-2. Besides, recent study finds that Bcl-2 is one of the
downstream genes of NF-κB [42]. As endogenous cells, IAPs
are apoptosis-inhibiting proteins, including cIAP1, cIAP2,
XIAP and survivin, etc. cIAP1 and cIAP2 can not only directly
inhibit the activation of caspases 3, caspases 7 and caspases 9
to inhibit cell apoptosis, but also induce the activation of NF-
κB [43]. On one hand, gene coding sequence of cIAP1 and
CIAP2 has κB site, so the activation of NF-κB can increase the
expression of cIAP1 and cIAP2. On the other hand, a large
number of expressed cIAP1 and cIAP2 can degrade IκB by
combining with TRAF, leading to the activation of NF-κB.
This forms the positive feedback loop mechanism, which
causes high expression of NF-κB, cIAP1 and cIAP2 in order to
prevent the apoptosis of tumor cells [44]. FLIP gene coding
sequence has κB site, so the high expression of activated NF-
κB protein in tumor cells can increase the transcriptional
expression of FLIP, which prevents tumor cells from
controlling by Fas-mediated apoptosis signals [45]. After
cross-linking with FasL or anti-Fas antibody, Fas can conduct
death signals into cells and induce cell apoptosis. Fas/FasL is
also one of CTL mechanisms. FasL is natural ligand of Fas and
exists on the surface of CTL. By combination between FasL
and CTL cells, the expression of Fas cells can lead to cell
apoptosis. As a result, Fas/FasL is a kind of immune defense
system, which is directly related to cancer cell apoptosis [46].

P53 protein is a key ingredient checkpoint in cell cycle G1.
The checkpoint of Gl can examine whether DNA is damaged
before synthesis. If there is damage, it needs to be repaired at
first. If repair fails, gene transcription which induces apoptosis
will be activated, such as activation of p21 gene [47]. Through
cyclin-dependent kinase (CDK), cell cycle stops at G1 and
begins apoptosis. Studies have shown that p53 gene mutation often happens in HCC. Besides, p53 mutation is closely related
to histological grading of liver cancer as well as attack and
metastasis of liver cancer. If the expression of P21 gene, an
important anti-cancer gene, is abnormal, the regulation of cell
proliferation and differentiation will be influenced, leading to
malignant tumor [48,49]. By depending on p53 way, p21 is
involved in cell cycle inhibition. Studies have shown that
within the cells, when the expression of p21 is low, the
combination between a molecule of p21 and cyclin-PCNA-CDK
is essential during cell cycle. When the expression of p21
is high, the combination between more molecules of p21 and
the above compounds inhibits cell cycle [50,51].

Genipin gentiobioside, geniposide, crocin 1, crocin 2 are all
substances with strong biological activity. Genipin gentiobioside can treat heart failure [52], while geniposide,
crocin 1, crocin 2 are confirmed to have anti-cancer effects,
such as on colon cancer and bladder cancer [12,53]. It has been
proved that gardenoside has certain effect on HepG2 liver
cancer cells cultured in vitro. The anti-cancer effect of Gardenia jasminoides also comes from these four kinds of
ingredients. The four kinds of ingredients can induce cancer
cell apoptosis through its influence on the expression of Bcl-2
and other kind of genes, which may be the main mechanism of
how Gardenia jasminoides achieve its anti-cancer effect. Gardenia jasminoides contains genipin gentiobioside,
geniposide, crocin 1and crocin 2, these chemical compositions
has the clear anticancer effects [54], geniposide injection fits
three compartment model in rat, its detention time in body was
short [54], pharmacokinetics of crocin 1and crocin 2 were also
determined in vitro, their anticancer effects were confirmed by
their cancer cells cytotoxic effect [55]. In this study, geniposide
content was highest, the key effect of Gardenia jasminoides might from geniposide.

Acknowledgments

This research project was supported by Science and
Technology Innovation Action Plan of Shanghai
(14495800400), Basic Research Project of Chongqing Frontier
and Application (cstc2014jcyjA1466) and Project of
Chongqing Social Science and Technology Innovation
(cstc2015shmszx120087), China.